Multivalent antibody contructs

ABSTRACT

The present invention relates to a multivalent F v  antibody construct having at least four variable domains which are linked with each over via the peptide linkers 1, 2 and 3. The invention also concerns expression plasmids which code for such an F v  antibody construct and a method of producing the F v  antibody constructs as well as their use.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 09/674,794,filed Aug. 21, 2001, which is a national phase filing of Application No.PCT/DE99/01350, which was filed with the Patent Cooperation Treaty onMay 5, 1999, and is entitled to priority of the German PatentApplication 198 19 846.9, filed May 5, 1998.

FIELD OF THE INVENTION

The present invention relates to multi-valent F_(v) antibody constructs,expression plasmids which code for them, and a method for producing theF_(v) antibody constructs as well as the use thereof.

BACKGROUND OF THE INVENTION

Natural antibodies are dimers and are therefore referred to as bivalent.They have four variable domains, namely two V_(H) domains and two V_(L)domains. The variable domains serve as binding sites for an antigen, abinding site being formed from a V_(H) domain and a V_(L) domain.Natural antibodies recognize one antigen each, so that they are alsoreferred to as monospecific. Furthermore, they also have constantdomains which add to the stability of the natural antibodies. On theother hand, they are also co-responsible for undesired immune responseswhich result when natural antibodies of various animal species areadministered mutually.

In order to avoid such immune responses, antibodies are constructedwhich lack the constant domains. In particular, these are antibodieswhich only comprise the variable domains. Such antibodies are designatedF_(v) antibody constructs. They are often available in the form ofsingle-chain monomers paired with one another.

However, it showed that F_(v) antibody constructs only have littlestability. Therefore, their usability for therapeutic purposes isstrongly limited.

Thus, it is the object of the present invention to provide an antibodyby means of which undesired immune responses can be avoided.Furthermore, it shall have a stability which makes it usable fortherapeutic uses.

According to the invention this is achieved by the subject mattersdefined in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a multivalent a multi-valent F_(v)antibody construct having at least four variable domains which arelinked with each other via the peptide linkers 1, 2 and 3. The inventionalso concerns expression plasmids which code for an F_(v) antibodyconstruct and a method of producing the F_(v) antibody constructs aswell as their use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genetic organization of an F_(v) antibody construct (A)according to the invention and schemes for forming a bivalent (B) ortetravalent F_(v) antibody construct (C) Ag: antigen; His₆: sixC-terminal histidine residues; stop: stop codon (TAA); V_(H) and V_(L):variable region of the heavy and light chains.

FIG. 2 shows the scheme for the construction of the plasmidspDISC3x19-LL and pDISC3x19-SL. c-myc: sequence coding for an epitopewhich is recognized by the antibody 9E1, His₆: sequence which codes forsix C-terminal histidine residues; PelB: signal peptide sequence of thebacterial pectate lyase (PelB leader); rbs: ribosome binding site; Stop:stop codon (TAA); V_(H) and V_(L): variable region of the heavy andlight chains.

FIG. 3 shows a diagram of the expression plasmid pDISC3x19-LL. 6×His:sequence which codes for six C-terminal histidine residues; bla: genewhich codes for β-lactamase responsible for ampicillin resistance; bp:base pairs; c-myc: sequence coding for an epitope which is recognized bythe 9E10antibody; ColE1: origin of the DNA replication; f1-IG:intergenic region of the bacteriophage f1; Lac P/O: wt lac-operonpromoter/operator; linker 1: sequence which codes for a GlyGly dipeptidelinking the V_(H) and V_(L) domains; linker 2: sequence coding for a(Gly₄Ser)₄ polypeptide which links the hybrid scFv fragments; Pel-Bleader: signal peptide sequence of the bacterial pectate lyase; rbs:ribosome binding site; V_(H) and V_(L): variable region of the heavy andlight chains.

FIG. 4 shows a diagram of the expression plasmid pDISC3x19-SL. 6×His:sequence which codes for six C-terminal histidine residues; bla: genewhich codes for β-lactamase which is responsible for the ampicillinresistance; bp: base pairs; c-myc: sequence coding for an epitoperecognized by the 9E10 antibody; ColE1: origin of DNA replication;f1-IG: intergenic region of the bacteriophage fl; Lac P/O: wt lac-operonpromoter/operator: linker 1: sequence which codes for a GlyGly dipeptidewhich links the V_(H) and V_(L) domains; linker 3: sequence which codesfor a GlyGlyProGlySer oligopeptide which links the hybrid scFvfragments; Pel-B leader: signal peptide sequence of the bacterialpectate lyase; rbs: ribosome binding site; V_(H) and V_(L): variableregion of the heavy and light chains.

FIG. 5 shows the nucleotide sequence and the amino acid sequence derivedtherefrom of the bivalent F_(v) antibody construct encoded by theexpression plasmid pDIS3x19-LL. c-myc epitope: sequence coding for anepitope which is recognized by the antibody 9E10; CDR: regiondetermining the complementarity; framework: framework region; His6 tail:sequence which codes for six C-terminal histidine residues; PelB leader:signal peptide sequence of the bacterial pectate lyase; RBS: ribosomebinding site; V_(H) and V_(L): variable region of the heavy and lightchains.

FIG. 6 shows the nucleotide sequence and the derived amino acid sequenceof the tetravalent F_(v) antibody construct encoded by the expressionplasmid pDISC3x19-SL. c-myc epitope: sequence coding for an epitopewhich is recognized by the 9E10 antibody; CDR: region determiningcomplementarity; framework: framework region; His6 tail: sequence codingfor the six C-terminal histidine residues; PelB leader: signal peptidesequence of the bacterial pectate lyase; RBS: ribosome binding site;V_(H) and V_(L): variable region of the heavy and light chains.

FIG. 7 shows the nucleotide sequence and the derived amino acid sequenceof a connection between a gene which codes for an α-factor leadersequence and a gene coding for the tetravalent F_(v) antibody constructin the Pichia expression plasmid pPIC-DISC-SL. Alpha-factor signal:leader peptide sequence of the Saccharomyces cerevisiae-α factorsecretion signal; V_(H): variable region of the heavy chain. Rhombsindicate the signal cleaving sites.

FIG. 8 shows the nucleotide sequence and the derived amino acid sequenceof a connection between a gene coding for an α-factor leader sequenceand a gene which codes for the bivalent F_(v) antibody construct in thePichia expression plasmid pPIC-DISC-LL. Alpha-factor signal: leaderpeptide sequence of the Saccharomyces cerevisiae-α-factor secretionsignal; V_(H): variable region of the heavy chain. Rhombs show thesignal cleaving sites.

FIG. 9 shows a diagram of the expression plasmid pDISC5-LL. 6×His:sequence coding for six C-terminal histidine residues; bla: gene whichcodes for β-lactamase responsible for ampicillin resistance; bp: basepairs; c-myc: sequence coding for an epitope which is recognized by the9E10 antibody; hok-sok: plasmid-stabilizing DNA locus; Lacd: gene whichcodes for the Lac represser; Lac P/O: wt lac-operon-promoter/operator,LacZ′: gene which codes for the α-peptide of β-galactosidase, linker 1:sequence which codes for a GlyGly dipeptide connecting the V_(H) andV_(L) domains; linker 2: sequence which codes for a (Gly₄Ser)₄polypeptide linking the hybrid scFv fragments; M13 IG: intergenic regionof the M13 bacteriophage; pBR322ori: origin of DNA replication; Pel-Bleader: signal peptide sequence of the bacterial pectate lyase; rbs:ribosome binding site which originates from the E. coli lacZ gene(lacZ), from the bacteriophage T7 gene 10 (T7g10) or from the E. coliskp gene (skp), -skp: gene which codes for the bacterial periplasmicfactor Skp/OmpH; tHP: strong transcription terminator; tIPP:transcription terminator; V_(H) and V_(L): variable region of the heavyand light chains.

FIG. 10 shows a diagram of the expression plasmid pDISC6-SL. 6×His:sequence which codes for six C-terminal histidine residues; bla: genewhich codes for β-lactamase responsible for ampicillin resistance; bp:base pairs: c-myc: sequence coding for an epitope which is recognized bythe 9E10 antibody; hok-sok: plasmid-stabilized DNA locus; Lacd: genewhich codes for the Lac represser; Lac P/O: wt lac-operonpromoter/operator; LacZ′: gene which codes for the α-peptide ofβ-galactosidase; linker 1: sequence which codes for a GlyGly dipeptidewhich links the V_(H) and V_(L) domains; linker 3: sequence which codesfor a GlyGlyProGlySer oligopeptide linking the hybrid scFv fragments:M13 IG: intergenic region of the M13 bacteriophage; pBR322ori: origin ofDNA replication; Pel-B leader: signal peptide sequence of the bacterialpectate lyase; rbs: ribosome binding site originating from the E. colilacZ gene (lacZ), from the bacteriophage TV gene 10 (T7g10) or from theE. coli skp gene (skp); skp: gene which codes for the bacterialperiplasmic factor Skp/OmpH; tHP: strong transcription terminator; tIPP:transcription terminator; V_(H) and V_(L): variable region of the heavyand light chains.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide an antibody bymeans of which undesired immune responses can be avoided. Furthermore,it shall have a stability which makes it useable for therapeutic use.

Therefore, the subject matter of the present invention relates to amulti-valent F_(v) antibody construct which has great stability. Such aconstruct is suitable for diagnostic and therapeutic purposes.

The present invention is based on the applicant's insights that thestability of an F_(v) antibody construct can be increased if it ispresent in the form of a single-chain dimer where the four variabledomains are linked with one another via three peptide linkers. Theapplicant also recognized that the F_(v) antibody construct folds withitself when the middle peptide linker has a length of about 10 to 30amino acids. The applicant also recognized that the F_(v) antibodyconstruct folds with other F_(v) antibody constructs when the middlepeptide linker has a length of about up to 10 amino acids so as toobtain a multimeric, i.e. multi-valent, F_(v) antibody construct. Theapplicant also realized that the F_(v) antibody construct can bemulti-specific.

According to the invention the applicant's insights are utilized toprovide a multi-valent F_(v) antibody construct which comprises at leastfour variable domains which are linked with one another via peptidelinkers 1, 2 and 3.

The expression “F_(v) antibody construct” refers to an antibody whichhas variable domains but no constant domains.

The expression “multivalent F_(v) antibody construct” refers to an F_(v)antibody which has several, but at least four, variable domains. This isachieved when the single-chain F_(v) antibody construct folds withitself so as to give four variable domains, or folds with othersingle-chain F_(v) antibody constructs. In the latter case, an F_(v)antibody construct is given which has 8, 12, 16, etc., variable domains.It is favorable for the F_(v) antibody construct to have four or eightvariable domains, i.e. it is bivalent or tetravalent (FIG. 1).Furthermore, the variable domains may be equal or differ from oneanother, so that the antibody construct recognizes one or severalantigens. The antibody construct preferably recognizes one or twoantigens, i.e. it is monospecific and bispecific, respectively. Examplesof such antigens are proteins CD19 and CD3.

The expression “peptide linkers 1, 3” refers to a peptide linker adaptedto link variable domains of an F_(v) antibody construct with oneanother. The peptide linker may contain any amino acids, the amino acidsglycine (G), serine (S) and proline (P) being preferred. The peptidelinkers 1 and 3 may be equal or differ from each other. Furthermore, thepeptide linker may have a length of about 0 to 10 amino acids. In theformer case, the peptide linker is only a peptide bond from the COOHresidue of one of the variable domains and the NH₂ residue of another ofthe variable domains. The peptide linker preferably comprises the aminoacid sequence GG.

The expression “peptide linker 2” refers to a peptide linker adapted tolink variable domains of an F_(v) antibody construct with one another.The peptide linker may contain any amino acids, the amino acids glycine(G), serine (S) and proline (P) being preferred. The peptide linker mayalso have a length of about 3 to 10 amino acids, in partiuclar 5 aminoacids, and most particularly the amino acid sequence GGPGS, which servesfor achieving that the single-chain F_(v) antibody construct folds withother single-chain F_(v) antibody constructs. The peptide linker canalso have a length of about 11 to 20 amino acids, in particular 15 to 20amino acids, and most particularly the amino acid sequence (G₄S)₄, (SEQID NO: 14) which serves for achieving that the single-chain F_(v)antibody construct folds with itself.

An F_(v) antibody construct according to the invention can be producedby common methods. A method is favorable in which DNAs coding for thepeptide linkers 1, 2 and 3 are ligated with DNAs coding for the fourvariable domains of an F_(v) antibody construct such that the peptidelinkers link the variable domains with one another and the resulting DNAmolecule is expressed in an expression plasmid. Reference is made toExamples 1 to 6. As to the expressions “F_(v) antibody construct” and“peptide linker” reference is made to the above explanations and, by wayof supplement, to Maniatis, T. et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory 1982.

DNAs which code for an F_(v) antibody construct according to theinvention also represent a subject matter of the present invention.Furthermore, expression plasmids which contain such DNAs also representa subject matter of the present invention. Preferred expression plasmidsare pDISC3x19-LL, pDISC3x19-SL, pPIC-DISC-LL, pPIC-DISC-SL, pDISC5-LLand pDISC6-SL. The first four were deposited with the DSMZ (DeutscheSammlungfur Mikroorganismen und Zellen) [German-type collection formicro-organisms and cells] on Apr. 30, 1998 under DSM 12150, DSM 12149,DSM 12152 and DSM 12151, respectively.

Another subject matter of the present invention relates to a kit,comprising:

-   -   (a) an F_(v) antibody construct according to the invention,        and/or    -   (b) an expression plasmid according to the invention, and    -   (c) conventional auxiliary agents, such as buffers, solvents and        controls.

One or several representatives of the individual components may bepresent.

The present invention provides a multi-valent F_(v) antibody constructwhere the variable dornains are linked with one another via peptidelinkers. Such an antibody construct distinguishes itself in that itcontains no parts which can lead to undesired immune reactions.Furthermore, it has great stability. It also enables to bind severalantigens simultaneously. Therefore, the F_(v) antibody constructaccording to the invention is perfectly adapted to be used not only fordiagnostic but also for therapeutic purposes. Such purposes can be seenas regards any disease, in particular a viral, bacterial or tumoraldisease.

The invention is explained by the below examples. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and the methods which are functionally equivalentare within the scope of the invention. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying drawings. Such modification are intended to fall within thescope of the appended claims.

EXAMPLES Example 1 Construction of the Plasmids pDISC3x19-LL andpDISC3x19-SL for the Expression of Bivalent, Bispecific and/orTetravalent, Bispecific F_(v) Antibody Constructs in Bacteria

The plasmids pHOG-αCD19 and pHOG-dmOKT3 which code for the scFvfragments derived from the hybridoma HD37 which is specific to humanCD19 (Kipriyanov et al., 1996, J. Immunol. Meth. 196, 51-62) and fromthe hybridoma OKT3 which is specific to human CDS (Kipriyanov et al.,1997, Protein Eng. 10, 445-453), respectively, were used for theconstruction of expression plasmids for a single-chain F_(v) antibodyconstruct. A PCR fragment 1 of the V_(H) domain of anti-CD19, followedby a segment which codes for a GlyGly linker, was produced using theprimers DP1,5′-TCACACAGAATTC-TTAGATCTATTAAAGAGGAGAAATTAACC (SEQ ID NO:1)and DP2,5-AGCACACGATATCACCGCCAAGCTTGGGTGTTGTTTTGGC (SEQ ID NO:2)(FIG.2). The PCR fragment 1 was cleaved by EcoRI and EcoRV and ligated withthe EcoRI/EcoRV-linearized plasmid pHOG-dmOKT3 so as to produce thevector pHOG19-3. The PCR fragment 2 of the V_(L) domain of anti-CD19,followed by a segment which codes for a c-myc epitope and ahexahistidinyl tail, was produced using the primers DPS,5′-AGCACACAAGCTTGGCGGTGATATCTTGCTCACCCAAACTCCA (SEQ ID NO:3) and DP4,5′-AGCACACTCTAGAGACACACAGATCTTTAGTGATGGTGATGGTGATGTGAGTTTAGG (SEQ IDNO:4). The PCR fragment 2 was cleaved by HindIII and XbaI and ligatedwith the HIndIII/XbaI-linearized plasmid pHOG-dmOKT3 so as to obtain thevector pHOG3-19 (FIG. 2). The gene coding for the hybrid scFv-3-19 inthe plasmid pHOG3-19 was amplified by means of PCR with the primersBi3sk, 5′-CAGCCGGCCATGGCGCAGGTGCAACTGCAGCAG (SEQ ID NO:5) and eitherLi-1,5′-TATATACTGCAGCTGCACCTGGCTACCACCACCACCGGAGCCGCCACCACCGCTACCACCGCCGCCAGAACCACCACCACCAGCGGCCGCAGCATCAGCCCG (SEQ ID NO:6) for the production of along flexible (Gly₄Ser)₄ inter-scFV linker (PCR fragment 3, FIG. 2) orLi-2,5′-TATATACTGCAGCTGCACCTGCGACCCTGGGCCACCAGCGGCCGCAGCATCAGCCCG (SEQID NO:7), for the production of a short rigid GGPGS linker (PCR fragment4, FIG. 2). The expression plasmids pDISC3x19-LL and pDISC3x19-SL wereconstructed by ligating the NcoI/PvuII restriction fragment frompHOG19-3, comprising the vector framework and the NcoI/PvuII-cleaved PCRfragments 3 and 4, respectively (FIGS. 3, 4). The complete nucleotideand protein sequences of the bivalent and tetravalent F_(v) antibodyconstructs are indicated in FIGS. 5 and 6, respectively.

Example 2 Construction of the Plasmids pPIC-DISC-LL and pPIC-DISC-SL forthe Expression of Bivalent, Bispecific and/or Tetravalent, BispecificF_(v) Antibody Constructs in Yeast

(A) Construction of pPIC-DISC-SL.

The vector pPICZαA (Invitrogen BV, Leek, Netherlands) for the expressionand secretion of recombinant proteins in the yeast Pichia pastoris wasused as a starting material. It contains a gene which codes for theSaccharomyces cerevisiae α-factor secretion signal, followed by apolylinker. The secretion of this vector is based on the dominantselectable marker, Zeocin™ which is bifunctional in both Pichia and E.coli. The gene which codes for the tetravalent F_(v) antibody construct(scDia-SL) was amplified by means of PCR by the template pDISC3x19-SLusing the primers 5-PIC,5′-CCGTGAATTCCAGGTGCAACTGCAGCAGTCTGGGGCTGAACTGGC and pSEXBn (SEQ IDNO:8). 5′-GGTCGACGTTAACCGACAAACAACAGATAAAACG (SEQ ID NO:9). Theresulting PCR product was cleaved by EcoRI and Xbal and ligated inEcoRI/XbaI-linearized pPICZαA. The expression plasmid pPIC-DISC-SL wasobtained. The nucleotide and protein sequences of the tetravalent F_(v)antibody construct are shown in FIG. 7.

(B) Construction of pPIC-DISC-LL.

The construction of pPIC-DISC-LL was carried out on the basis of pPICZαA(Invitrogen-BV, Leek, Netherlands) and pDISC3x19-LL (FIG. 3). Theplasmid-DNA pPICZαA was cleaved by EcoRI. The overhanging 5′-ends werefilled using a Klenow fragment of the E. coli DNA polymerase I. Theresulting DNA was cleaved by Xbal, and the large fragment comprising thepPIC vector was isolated. Analogous thereto the DNA of pDISC3x19-LL wascleaved by NcoI and treated with a Klenow fragment. Following thecleavage using Xbal a small fragment, comprising a gene coding for thebivalent F_(v) antibody, was isolated. Its ligation with a pPIC-derivedvector-DNA resulted in the plasmid pPIC-DISC-LL. The nucleotide andprotein sequences of the bivalent F_(v) antibody construct are shown inFIG. 8.

Example 3 Expression of the Tetravalent and/or Bivalent F_(v) AntibodyConstruct in Bacteria

E. coli XLI-blue cells (Strategene, La Jolla, Calif.) which had beentransformed with the expression plasmids pDISC3x19-LL and pDISC3x19-SL,respectively, were cultured overnight in 2×YT medium with 50/μg/mlampicillin and 100 mM glucose (2×YT_(Ga)) at 37° C. 1:50 dilutions ofthe overnight cultures in 2×YT_(GA) were cultured as flask cultures at37° C. while shaking with 200 rpm. When the cultures had reached anOD₆₀₀ value of 0.8, the bacteria were pelleted by 10-minutecentrifugation with 1500 g at 20° C. and resuspended in the same volumeof a fresh 2×YT medium containing 50 μg/ml ampicillin and 0.4 Msaccharose. IPTG was added up to a final concentration of 0.1 mM, andthe growth was continued at room temperature (20-22° C.) for 18-20 h.The cells were harvested by 10-minute centrifugation with 5000 g at 4°C. The culture supernatant was held back and stored on ice. In order toisolate the soluble periplasmic proteins, the pelleted bacteria wereresuspended in 5% of the initial volume of ice-cold 50 mM Tris-HCl, 20%saccharose, 1 mM EDTA, pH 8.0. Following 1 hour of incubation on icewith occasional stirring the spheroplasts were centrifuged with 30,000 gat 4° C. for 30 minutes, the soluble periplasmic extract being obtainedas supernatant and the spheroplasts with the insoluble periplasmicmaterial being obtained as pellet. The culture supernatant and thesoluble periplasmic extract were combined and clarified by furthercentrifugation (30,000 g, 4° C., 40 min.). The recombinant product wasconcentrated by ammonium sulfate precipitation (final concentration 70%saturation). The protein precipitate was obtained by centrifugation(10,000 g, 4° C., 40 min.) and dissolved in 10% of the initial volume of50 mM Tris-HCl, 1 M NaCl, pH 7.0. An immobilized metal affinitychromatography (IMAC) was carried out at 4° C. using a 5 ml column ofchelating sepharose (Pharmacia) which was charged with Cu²⁺ and had beenequilibrated with 50 mM Tris-HCl, 1 M NaCl, pH 7.0 (starting buffer).The sample was loaded by passing it over the column. It was then washedwith twenty column volumes of starting buffer, followed by startingbuffer with 50 mM imidazole until the absorption at 280 nm of theeffluent was at a minimum (about thirty column volumes). The absorbedmaterial was eluted with 50 mM Tris-HCl, 1 M NaCl, 250 mM imidazole, pH7.0.

The protein concentrations were determined with the Bradford dye bindingtest (1976, Anal. Biochem. 72, 248-254) using the Bio-Rad (Munich,Germany) protein assay kit. The concentrations of the purifiedtetravalent and bivalent F_(v) antibody constructs were determined fromthe A₂₈₀ values using the extinction coefficients ε^(1 mg/ml)=1.96 and1.93, respectively.

Example 4 Expression of the Tetravalent and/or Bivalent AntibodyConstruct in the Yeast Pichia pastoris

Competent P. pastoris GS155 cells (Invitrogen) were electroporated inthe presence of 10 μg plasmid-DNA of pPICDISC-LL and pPIC-DISC-SL,respectively, which had been linearized with SacI. The transformantswere selected for 3 days at 30° C. on YPD plates containing 100 μg/mlZeocin™. The clones which secreted the bivalent and/or tetravalent F_(v)antibody constructs were selected by plate screening using ananti-c-myc-mAk 9E10 (1C Chemikalien, Ismaning, Germany).

For the expression of the bivalent F_(v) antibody constructs andtetravalent F_(v) antibody constructs, respectively, the clones werecultured in YPD medium in shaking flasks for 2 days at 30° C. withstirring. The cells were centrifuged resuspended in the same volume ofthe medium containing methanol and incubated for another 3 days at 30°C. with stirring. The supernatants were obtained after thecentrifugation. The recombinant product was isolated by ammonium sulfateprecipitation, followed by IMAC as described above.

Example 5 Characterization of the Tetravalent F_(v) Antibody Constructand Bivalent F_(v) Antibody Construct, Respectively

(A) Size Exclusion Chromatography.

An analytical gel filtration of the F_(v) antibody constructs wascarried out in PBS using a superdex 200-HR10/30 column (Pharmacia). Thesample volume and the flow rate were 200 μl/min and 0.5 ml/min,respectively. The column was calibrated with high-molecular andlow-molecular gel filtration calibration kits (Pharrmacia).

(B) Flow Cytometry.

The human CD3⁺/CD19⁻-acute T-cell leukemia line Jurkat and theCD19⁺/CD3⁻B-cell line JOK-1 were used for flow cytometrie. 5×10⁵ cellsin 50 μl RPMI 1640 medium (GIBCO BRL, Eggestein, Germany) which wassupplemented with 10% PCS and 0.1% sodium azide (referred to as completemedium) were incubated with 100 μl of the F_(v) antibody preparationsfor 45 minutes on ice. After washing using the complete medium the cellswere incubated with 100 μl 10 μg/ml anti-cmyc-Mak 9E10 (1C Chemikalien)in the same buffer for 45 minon ice. After a second wash cycle, thecells were incubated with 100 μl of the FITC-labeled goat-anti-mouse-IgG(GIBCO BRL) under the same conditions as before. The cells were thenwashed again and resuspended in 100 μl 1 μg/ml propidium iodide solution(Sigma, Deisenhofen, Germany) in complete medium with the exclusion ofdead cells. The relative fluorescence of the stained cells was measuredusing a FACScan flow cytometer (Becton Dickinson, Mountain View,Calif.).

(C) Cytotoxicity Test.

The GDI9-expressing Burkitt lymphoma cell line Raji and Namalwa wereused as target cells. The cells were incubated in RPMI 1640 (GIBCO BRL)which was supplemented with 10% heat-inactivated PCS (GIBCO BRL), 2 mMglutamine and 1 mM pyruvate, at 37° C. in a dampened atmosphere with7.5% CO₂. The cytotoxic T-cell tests were carried out in RPMI-1640medium supplemented with 10% FCS, 10 mM HEPES, 2 mM glutamine, 1 mMpyruvate and 0.05 mM 2-ME. The cytotoxic activity was evaluated using astandard [⁵¹Cr] release test; 2×10⁶ target cells were labeled with 200μCi Na [⁵¹Cr] O₄ (Amersham-Buchler, Braunschweig, Germany) and washed 4times and then resuspended in medium in a concentration of 2×10⁵/ml. Theeffector cells were adjusted to a concentration of 5×10⁶/ml. Increasingamounts of CTLs in 100 μl were titrated to 10⁴ target cells/well orcavity in 50 μl. 50 μl antibodies were added to each well. The entiretest was prepared three times and incubated at 37° C. for 4 h. 100 μl ofthe supernatant were collected and tested for [⁵¹Cr] release in a gammacounter (Cobra Auto Gamma; Canberra Packard, Dreieich, Germany). Themaximum release was determined by incubation of the target cells in 10%SDS, and the spontaneous release was determined by incubation of thecells in medium alone. The specific lysis (%) was calculated as:(experimental release−spontaneous release)/(maximum release−spontaneousrelease)×100.

Example 6 Construction of the Plasmids pDISC5-LL and pDISC5-SL for theExpression of Bivalent, Bispecific and/or Tetravalent, Bispecific F_(v)Antibody Constructs in Bacteria by High Cell Density Fermentation

Expression vectors were prepared which contained the hok/sokplasmid-free cell suicide system and a gene which codes for the Skp/OmpHperiplasmic factor for a greater production of recombinant antibodies.The skp gene was amplified by PCR using the primers skp-1,5′-CGA ATT CTTAAG ATA AGA AGG AGT TTA TTG TGA AAA AGT GGT TAT TAG CTG CAG G (SEQ IDNO: 10) and skp-2,5′-CGA ATT AAG CTT CAT TAT TTA ACC TGT TTC AGT ACG TCGG (SEQ ID NO:11) using the plasmid pGAH317 (Hoick and Kleppe, 1988, Gene67, 117-124). The resulting PCR fragment was cleaved by AflII andHindIII and inserted in the AflII/HindIII-linearized plasmid pHKK (Hornet al., 1996, Appl. Microbiol. Biotechnol. 46, 524-532) so as to obtainthe vector pSKK. The genes obtained in the plasmids pDISC3x19-LL andpDISC3x19-SL and coding for the scFv antibody constructs were amplifiedby means of the primers fe-1,5′-CGA ATT TCT AGA TAA GAA GGA GAA ATT AACCAT GAA ATA CC (SEQ ID NO:12) and fe-2,5′-CGA ATT CTT AAG CTA TTA GTGATG GTG ATG GTG ATG TGA G (SEQ ID NO:13). The XbaI/AflII-cleaved PCRfragments were inserted in pSKK before the skp insert so as to obtainthe expression plasmids pDISC5-LL and pDISC6-SL, respectively, whichcontain tri-cistronic operons under the control of the lacpromoter/operator system (FIGS. 9, 10).

1. A bivalent monomeric F_(v) antibody formed by one single-chain F_(v)monomer having four variable domains, wherein said four variable domainsare V_(H)-A, V_(L)-B, V_(H)-B and V_(L)-A, wherein V_(H)-A and V_(L)-Aare V_(H) and V_(L) domains of an antibody specific for antigen A,respectively, and V_(H)-B and V_(L)-B are V_(H) and V_(L) domains of anantibody specific for antigen B, respectively; V_(H)-A is linked toV_(L)-B by peptide linker 1, V_(L)-B is linked to V_(H)-B by peptidelinker 2 and V_(H)-B is linked to V_(L)-A by peptide linker 3; saidpeptide linker 1 and said peptide linker 3 are a peptide bond or have 1to about 10 amino acids; and said peptide linker 2 has about 10 to about30 amino acids; V_(H)-A and V_(L)-A associate intramolecularly to form abinding site for antigen A; and V_(L)-B and V_(H)-B associateintramolecularly to form a binding site for antigen B.
 2. The F_(v)antibody of claim 1, wherein said peptide linker 2 have 11 to about 20amino acids.
 3. The F_(v) antibody of claim 1, wherein said peptidelinker 2 have 15 to 20 amino acids.
 4. The Fv antibody of claim 1,wherein said peptide linker 2 comprises the amino acid sequence GGPGS.5. The F_(v) antibody of claim 1, wherein said peptide linker 2 is(G₄S)₄.
 6. The F_(v) antibody of claim 1, wherein said peptide linker 1and said peptide linker 3 have the amino acid sequence GG.
 7. The F_(v)antibody of claim 1, wherein said peptide linker 1 and said peptidelinker 3 have the same amino acid sequence.
 8. The F_(v) antibody ofclaim 1, wherein said antibody is monospecific.
 9. The F_(v) antibody ofclaim 1, wherein said antibody is bispecific.
 10. The F_(v) antibody ofclaim 1, wherein said antibody is specific for an antigen selected fromthe group consisting of CD3 and CD19.
 11. The F_(v) antibody of claim 9,wherein said antibody is specific for CD3 and CD19.
 12. A compositioncomprising said F_(v) antibody of claim 1 for diagnosis and/or treatmentof diseases.
 13. A method for producing said F_(v) antibody of claim 1,comprising the steps of: ligating DNAs encoding said four variabledomains V_(H)-A, V_(L)-B, V_(H)-B and V_(L)-A of said single chain F_(v)monomer with DNAs encoding peptide linker 1, peptide linker 2 andpeptide linker 3 to produce a DNA encoding said single-chain F_(v)monomer; and cloning the DNA encoding said single-chain F_(v) monomerconstruct into an expression plasmid to produce an expression vector forsaid single-chain F_(v) monomer; transforming a host cell with theexpression plasmid for said monomer single-chain Fv-monomer; andcultivating the host cell under conditions that said single-chain F_(v)monomer is expressed.
 14. The method of claim 13, wherein the expressionplasmid for said single-chain F_(v) monomer is selected from the groupconsisting of pDisc3x19-LL, pPIC-DISC-LL and pDISC5-LL as deposited withDSM.